1. Field of the Invention
The present invention provides a novel treatment for senile dementia (Alzheimer's Type), comprising administering an anti-complement protein to a patient in need of such treatment in an amount sufficient to inhibit the complement cascade and thereby inhibit the production of amyloid plaques in the brain of the patient.
2. Description of the Related Art
Alzheimer's Disease (AD) is a slowly debilitating neurodegenerative chronic illness that may progress for a decade or longer before death ensues. The disease often strikes later in life. This is evidenced by the fact that half of those over the age of 80 years are afflicted with the disorder. At present, it is the fourth leading cause of adult deaths in the US alone, at an annual cost of approximately $100 billion. As the longevity of the world's population increases, this disease will become an even greater problem unless a better understanding of the disease process and its management is achieved.
Alois Alzheimer is credited with being the first to diagnose what is now known as Alzheimer's disease (AD). In 1906, Alzheimer reported a case of what he termed “presenile dementia” in a 51 year old patient at a psychiatric meeting in Southwest Germany. He recognized certain characteristics that he felt differentiated it from the usual diagnosis of dementia. First was the early onset of the disease in an otherwise healthy young woman. More importantly, however, were the histological changes he found in sections of brain tissue from the patient. Alzheimer described seeing amyloid (starch-like) plaques and coarse-fibered proliferations of neurofibrils under the microscope. Several other researchers in years following reported similar findings of presenile dementia, and in 1910 a textbook of psychiatric disorders defined this form of dementia as “Alzheimer's disease.” The eponym was adopted in the literature and became the standard. It is perhaps fitting, since Alzheimer's original observations are still the main criteria of diagnosis for the disease.
The plaques and neurofibrils described by Alzheimer, which are today called senile plaques and neurofibrillary tangles (NFT), are used as a definitive diagnosis of AD (FIG. 1A (Palmert, M. R. et al. (1996) Science 24:11080–11084). The plaques and tangles are seen primarily in the hippocampus, amygdale, and the cerebral cortex (Van Broeckhoven, C. L. (1995) Eur. Neurol. 35:8–19). Evidence for either a molecular or immunological disease origin may be found in the plaques and tangles, depending upon a researcher's point of reference. From a molecular perspective, the initial identification of specific mutations within the amyloid precursor protein (APP) (Schellenberg, G. D. et al. (1991) 49:511–517) and the presence of Aβ (a derivative of APP) in plaques points to a unique protein cause for AD.
Several other protein players have since entered the AD arena. These include the already mentioned major constituent of NFTs, tau, as well as three recent additions. Presenilins 1 and 2 are integral membrane proteins coded for on different chromosomes, that when mutated are responsible for up to 90% of the cases of autosomal dominant early-onset familial Alzheimer's disease (FAD) (Thinakaran, G. et al. (1996) Neuron 17(1):181–190). Although FAD accounts for only 10%, of all cases of AD, there is evidence of an interaction between the presenilins and APP (Weidemann, A. et al. (1997)Nat. Med. 3(3):328–332). Therefore, even normal forms of the proteins may play a role in the far more common sporadic AD. Finally, a specific isoform of apolipoprotein E (apoE), apoE4, has been shown to be a strong genetic risk factor for AD (Stritmatter, W. J. et al. (1993) Proc. Natl. Acad. Sci. USA 90:8098–8102). People carrying two copies of the of the E4 isoform have a statistically greater risk of developing late-onset AD and in vitro experiments have shown that apoE is capable of binding to Aβ (Stritmatter, W. J. et al. (1994) Proc. Natl. Acad. Sci. USA 91:11183–11186).
The histopathological investigations into AD also point to the immune response having an important role in disease progression. The presence of activated microglia, reactive astrocytes, acute phase proteins, and complement factors within and around neuritic plaques are all signs of in inflammatory response. It is known that APP is capable of binding specifically to C1q, which can trigger activation of the classical pathway of the complement cascade in an antibody-independent manner (Webster, S. et al. (1995) Biochem. Biophys. Res. Comm. 217:869–875). Deposition of complexes and formation of immunomodulators by the cascade have been credited with activating the microglia—macrophages of the brain—which in turn cause a progression and maintenance of the inflammation. Local tissue destruction follows, along with a further persistence and increase in inflammation.
Histological and Immunohistopathological Features of AD
The plaques are the most distinctive feature found in the brain tissue of patients with AD. These characteristic plaques are composed of an aggregation of the Aβ peptide, which is a by-product of APP metabolism. Morphologically distinct varieties of Aβ deposits have been described from conventional silver staining of histological sections:
Early lesions, referred to as diffuse plaques, are formed from Aβ deposits but are not associated with dystrophic neurites. Diffuse plaques are found in nondemented persons over the age of 65 years and are immunoreactive with a variety of different anti-AP antibodies. However, they generally are not stained with dyes like Congo red and thioflavin S.
In sharp contrast, amyloid plaques (also referred to as neuritic plaques because they contain cells and subcellular components such as astrocytic processes, dystrophic neurites, microglia, neurons with or without neurofibrillary tangles and proteins such as complement components, apolipoprotein E and alpha-1-chymotrypsin) develop at later stages of AD. As seen in the autopsied brain tissue in FIG. 1, they appear dark in the center when stained with silver, and also stain with both Congo red and thioflavin S.
Recently, new lesions known as “AMY” plaques have been reported. This third type of plaque is similar to the amyloid plaques but have no central amyloid core (Schmidt, M. L. et al. (1995) Am. J. Pathol 147:503–515.
Besides plaques, the intracellular neurofibrillary tangles are often also characteristic of AD brain tissue. The plaques and neurofibrillary tangles are the primary diagnostic features of AD. However, immunohistochemical staining is becoming more useful as additional antibodies specific to plaque components are developed. An immunodominant region of APP has been localized to the C-terminal tail. This region of the processed APP had been postulated to remain intracellular and was recently shown to accumulate specifically in the neuronal cells of the hippocampus and amygdale of AD patients, but not in similar tissue of the normal age-matched patients (Kotwal, G. J. et al. (1997) Soc. Neurosci. Abstr. 22:502). As shown in FIG. 1B, a high titer antibody to the immunodominant region can give rise to specific intracellular immunohistochemical staining in the amygdale, which may someday find routine usage to confirm diagnosis in conjunction with clinical history and silver staining. Additionally, the presence of the C-terminus demonstrates that the C-terminal tail of APP accumulates intracellularly in neural tissue of those suffering from AD.
Molecular Genetics of AD
There are several critical molecules their directly or indirectly contribute to the disease state in AD (Selkoe, D. J. (1997) Science 275:630–631). These molecules, their chromosomal locations, and how they may impact AD are summarized in Table 1.
TABLE 1Major proteins implicated in Alzheimer's DiseaseEffect on AβSporadic v.ProteinChromosomeGene DefectAge of OnsetProductionFamilialAPP21Mutations in the50sIncreasedSporadic/FamilialC-terminusProductionApoE419Polymorphisms>60IncreasedSporadicdensity of AβplaquesPresenilin 114Mutations40s and 50sIncreasedFamilialproductionPresenilin 21Mutations50sIncreasedFamilialproductionTau17None foundUnknownNo knownSporadic?influence onAβ synthesisThe genetic defects in the critical molecules identified to date lead to increased biosynthesis increased aggregation, or decreased clearance of Aβ peptides. Thus, Aβ accumulation is a necessary step in the pathogenesis of AD, but does not account for all the pathogenesis of AD. The gene that is implicated as a risk factor in late-onset sporadic AD and associated with increased risk for AD is the lipid carrier, apolipoprotein E (apoE) epsilon 4 allele (Stritmatter, W. J. & Roses, A. D. (1996) Ann. Rev. Neurosci. 19:53). It is suggested that apoE4 is less efficient at cell repair than the alleles E2 and E3. The apoE4 is thought not only to contribute to the formation of β-amyloid plaques, but also to account for the neurofibrillary tangles. It has been suggested that apoE may bind to Aβ and after exiting astrocytes may get trapped into low-density lipoprotein (LDL)-related proteins present on the surface of neurites of apoptotic neurons. ApoE is further suggested to provide protective binding to the tau protein, thus preventing homodimerization. The homodimerization creates paired helical filament formation, which in turn forms the neurofibrillary tangles. In addition, it has been proposed that the alleles of apoE (except E4) contribute to the formation of stabilized cross-bridges on microtubules, and that in the absence of the tau protein microtubules cannot assemble and so the shape and synaptic integrity of cells are not maintained. Since apoE4 has a high affinity for Aβ and the lowest among the isoforms for tau it cannot prevent the formation of neurofibrillary tangles and does not allow microtubule assembly, thus contributing to some of the pathogenesis of AD (Roses, A. D. (1997) Hosp. Pract. 32(7):51–63).
The most recent group of proteins discovered to have an involvement in AD are the two presenilins (PS1 and PS2). Point mutations in these proteins are sufficient to cause early-onset FAD. The roughly 30 mutations in PSI and 2 in PS2 account for at least 50% of the early-onset FAD cases. These proteins are highly conserved with each other.
Structure, Processing, and Interaction of Critical Molecules
APP is an integral membrane glycoprotein with a large extracellular amino terminus, a single passage through the cell membrane, and a short carboxy-terminal tail, as illustrated in FIG. 2. It is expressed ubiquitously throughout the body and is found in several tissue-specific isoforms due to variant splicing. The largest isoform (APP770) contains a putative kunitz protease inhibitor (KPI); however, the form found predominantly in neuronal tissue, APP695 does not contain the KPI region. The normal functions of APP have been proposed to include action as a cell receptor, involvement in cell-cell interactions, and inhibition of proteases (KPI), among others (Selkoe, D. J. (1997) Science 275:630–631).
APP contains a signal peptide on its N-terminus and is membrane-anchored, so it is processed through the secretory pathway after translation (see FIG. 3). This pathway begins with cotranslational translocation across the membrane of the endoplasmic membrane (ER) into its lumen, with anchorage in the membrane occurring at the transmembrane domain. Processing of the protein to maturity, including glycosylation and sulfation, continues through the ER and into the Golgi network (Adams, C. (1997) Gerontology 43:8–19).
Proteolytic cleavage of APP can also occur as part of its processing through the secretary pathway. Somewhere between the trans-Golgi network and its localization on the cell surface, APP is cleaved at a region on the extracellular side of APP near the membrane in the region that could otherwise form Aβ (Sisodia, S. S. et al. (1990) Science 248:492–495), thus prohibiting its formation. The membrane-bound endoprotease responsible for the cleavage has been dubbed α-secretase and appears to require for its specificity no more than an α-helical conformation and a specified distance of 12–13 residues from the membrane (Sisodia, S. S. (1992) Proc. Nat. Acad. Sci. USA 89:6075–6079). If cleavage occurs, the soluble amino terminus is released into the solvent, while the carboxyl end remains membrane bound, presumably being degraded. If α-secretase does not hydrolyze APP, then the intact protein may be rapidly reinternalized via a clathrin-mediated endosomal pathway, where it may be either recycled to the surface through an early endosome or fused with a lysosome for degradation (Selkoe, D. J. (1996) J. Biol. Chem. 271:18295–18298).
Aβ is likely to be formed during this process, probably in the early endosome, due to proteolytic cleavage by enzyme(s) called β-secretase and γ-secretase (see FIG. 2). Recycling to the surface would result in release of Aβ into the extracellular medium. It has been demonstrated that Aβ production requires APP to be membrane bound and localized to a slightly acidic vesicle, such as the early endosome or the late trans-Golgi (Selkoe, D. J. (1996) Ann. N.Y. Acad. Sci. 777:57–64). The entire processing pathway is tightly controlled. For instance, upregulation of the protein kinase C (PKC) pathway by muscarinic receptors results in an increase in the α-secretase cleavage and a decrease in Aβ production. Serum levels of ligands for these receptors, such as acetylcholine and interleukin-1 (IL-1), have been reported to be abnormal in AD patients (Buxbaum, J. D. & Greengard, P. (1996) Ann. N.Y. Acad. Sci. 777:327–331). An intracellular increase in calcium will also upregulate α-secretase activity (Adams, C. (1997) Gerontology 43:8–19).
It is known that mutating APP, such as occurs in some cases of early-onset FAD, is sufficient to cause AD. The presence of Aβ in diffuse plaques (precluding symptom onset), its ability to form insoluble filaments in vitro, trisomy 21 in Down's syndrome (patients develop early-onset AD), the exhibited direct neurotoxicity, and the interaction with numerous molecules believed to be involved with AD including immune molecules, all point to APP's, and specifically Aβ's, pivotal role in AD pathogenesis (Spillantini, M. G. et al. (1996) Acta Neuropath. (Berl.) 92:4248).
One likely mechanism proposed (Stritmatter, W. J. & Roses, A. D. (1996) Ann. Rev. Neurosci. 19:53) is that either an increase in Aβ production or a decrease in clearance caused by a variety of different genetic, molecular, or environmental conditions results in Aβ accumulation into fibrils and then diffuse plaques. These plaques activate an inflammatory response (see below and FIG. 4), which can cause local cellular damage, in turn creating more inflammation. Alternatively, or together, the Aβ in the plaques may be directly toxic to neurons, resulting in a similar outcome. The cell damage could result in metabolic changes, which might explain the production of the NFTs seen associated with AD. Persistence of inflammation would result in an ever-spreading synaptic loss and eventual cell death, up to and beyond clinical symptoms of impairment.
A final proposed role of APP in AD involves its C-terminal region. It has been demonstrated that a specific region of the APP terminus (amino acids 657–676) is able to specifically bind and activate heterotrimeric G proteins (see FIG. 2) (Selkoe, D. J. (1996) J. Biol. Chem. 271:18295–18298). More specifically, a generated peptide corresponding to APP657-676 will bind only with the Gα0 subset of G proteins, and it binds with even greater affinity if the transmembrane region is included (Dragunow, M. & Preston, K. (1995) Brain Res. Rev. 2:11–28). This suggests that APP acts as a Go-coupled signal receptor. This theory is supported by experiments using a monoclonal antibody generated to the extracellular domain of APP. Application of this antibody to APP695 increased binding of the C-terminus G0, but not to other cellular heterotrimeric G proteins (Lassmann, H. et al. (1995) Acta Neuropath. (Berl.) 92:42–48). Activation of G proteins can result in a signal cascade which ends with apoptotic cell death. It may be that the APP C-terminus could be pathogenic in apoptotic manner, if it accumulates past a critical threshold within cells.
The other important component of AD histopathology is the neurofibrillary tangles (NFTs). There is good evidence that NFTs play an important role in the initiation of AD pathology. This evidence focuses on the microtubule associated protein, tau. When functioning properly, tau binds microtubules and promotes their stable polymerization into fibrils within the cell (Roses, A. D. (1997) Hosp. Pract. 32(7):51–63). When tau becomes hyperphosphorylated, it is no longer able to bind microtubules, which causes them to become destabilized, thus resulting in disruption of cellular trafficking and compromises in cytoskeletal integrity. Tau hyperphosphorylation results from an impaired ability to remove phosphates. The microtubule destabilization has been proposed to disrupt axonal transport which causes dying back of axons, impairing synaptic transmission (Peskind, E. R. (1996) J. Clin. Psychiatry 57 (Suppl. 14): 5–8). Such a mechanism of AD pathogenesis would not require an interaction with Aβ and therefore would not necessitate plaque formation. Indeed, comparison of a recently discovered presenile dementia lacking plaques but containing tangles shows tau forms and NFTs to be identical to those in AD according to several different analytical methods (Spillantini, M. G. et al. (1996) Acta Neuropath. (Berl.) 92:42–48).
Hyperphosphorylated tau binds itself, forming long filaments (PHFs), which accumulate intracellularly to form the recognized NFTs. It his been shown that carbamoylation or glycation of cationic tau residues will result in NFTs like those seen in AD. These cationic sites, particularly lysine residues, appear to be important for microtubule binding. Therefore, the phosphorylation of tau probably blocks the normal binding sites of tau to microtubules, preventing interaction (Farias, G. et al., (1997) Mol. Cell. Biochem. 168:59–66). NFTs may themselves be pathogenic if they accumulate to such an extent within the cell that they impair normal cellular processes. Also, it has been postulated that isoforms of apoE may interact with tau as well and inhibit or enhance NFT formation (discussed above).
As discussed in detail earlier, ApoE plays a role in at least sporadic AD, and perhaps FAD as well. In particular, the ApoE4 isoform increases the likelihood and decreases the age of onset of AD (Corder, E. H. et al. (1993) Science 261:921–923). ApoE is a 34-kDa protein found circulating throughout the body as well as the central nervous system. In the brain, ApoE scavenges lipid from degenerating neurons and redistributes it to branching neurites via uptake by the low-density lipoprotein (LDL) or LDL-related protein (LRP) receptors (Mahley, R. W. et al. (1996) Ann. N.Y. Acad. Sci. 777:139–145). ApoE also binds Aβ, and so along with its receptor may be a means of mopping up extracellular Aβ (Rebeck, G. W. (1995) Ann. Neurol. 37:211–217).
Presenilins 1 and 2 (PS1, PS2) are also involved in AD. PS1 has been shown to be membrane-anchored and contain eight membrane-spanning regions. The amino and carboxyl termini are both located on the cytoplasmic side of the membrane, along with a large hydrophilic loop between transmembrane domains six and seven. This loop is proteolytically cleaved during maturation of the protein to produce an approximately 25–28 kDa N-terminal and an approximately 16–19 kDa C-terminal protein. The shape is hypothesized to be a barrel within the membrane, with the loop acting as a gate (Rohan de Silva, H. A., & Patel, A. J. (1997) Neuroreport 8(8):i–xii). The mature form has been shown to localize primarily to the perinuclear membrane regions (ER and Golgi), with a small percentage in the surface membrane (Tanzi, R. E. et al. (1996) Alzheimer's Dis. Rev. 1:190–198).
The presenilins show close homology to two proteins of Caenorhabditis elegans, SPE4 and SEL12. SPE4 mediates the docking of a Golgi-derived organelle which stores and transports polypeptides (Takeshima, A. et al. (1996) Biochem. Biophys. Res. Comm. 227:423–426). This suggests a role in protein trafficking through the secretary pathway for the presenilins. SEL12 facilitates signaling by the Notch family of receptors (Beyreuther, K. & Masters, C. L. (1997) Nature Med. 3:723–725). These receptors are involved in determination of cell fate during development. This may indicate a role in cell signaling for the presenilins, or perhaps SEL 12 controls transport of second messengers between the nuclear and surface membrane. The latter possibility would again suggest a role for the presenilins in protein trafficking.
A role in cell signaling or protein trafficking relates to AD pathogenesis in the following manner. It is known that mutations in either of the presenilins result in an increase in the expression of Aβ42 without an increase in APP expression (Citron, M. et al. (1997) Nature Med. 3:67–72). Also, immunoprecipitation of coexpressed PS2 and APP in cell culture results in complexes of PS2/immature APP (Weidemann, A. et al. (1997)Nat. Med. 3(3):328–332). Together, these two pieces of evidence, along with homology and localization studies, suggest that presenilins regulate trafficking and processing of APP through the secretary pathway. When mutations occur in presenilins, it is likely that conformational changes induce a change of function or a new function with regard to APP processing, which results in an increase in the amount of Aβ produced.
Complement-Mediated Inflammatory Response in AD
The neuritic/amyloid plaques which are the hallmark of AD consist of aggregated Aβ, along with a number of complement components and complement control proteins such as C1 inhibitor and other noncomplement proteins. In addition, the neuritic plaques are associated with damaged (dystrophic) neuronal processes, activated microglia, and reactive astrocytes.
An area of intense research has been the antibody-independent activation of complement by nonfibrillar Aβ, the form found in diffuse plaques in the brain. An even more potent activator of complement is the β-pleated sheet aggregated Aβ that forms the fibrils found in neuritic plaques (Webster, S. et al. (1996) Mol. Immunol. 33:29). Both the classical complement pathway (CCP) and the alternate complement pathway (ACP) are activated by Aβ, leading to the formation of ester linked Aβ/C3 complexes, generation of a potent proinflammatory response due to products C5a and C5b-9, and formation of membrane attack complexes (MACS) (id.). This local inflammatory response results in the development of an intensely neurotoxic environment due to the influx and activation of glial cells, and damage to neurons near the neuritic plaques.
Specific interaction domains of the Aβ peptide involved in binding to C1q have been reported by Tenner's group. Amino acid residues 4–11 of Aβ have been identified as critical in the binding to C1q, the subunit of C1 that is involved in the activation of the classical complement pathway. Residue number 7, an aspartic acid in Aβ, is believed to be a very critical site for this protein to interact with C1q (Velazquez, P. et al. (1997) Nature Med. 3:77–79). Because of the activation of complement and the triggering of signal transduction processes in the classic injury cascade in response to the Aβ; it has been suggested that an amplified Aβ cascade is set off which converts an acute-phase injury response into a chronic one (Cotman, C. W. et al. (1996) Neurobiol. Aging 17:723–731). This chronic response is supposedly maintained by continuous stimulation and injury and may be an underlying cause for neuronal dysfunction and progressive degeneration.
Due to the clear involvement of complement in the inflammatory response in AD, targeting the complement system would be expected to slow or stop the progression of AD without affecting the beneficial effects of the normal protective functions of the body's first line of defense. In addition, active regions of several of the microbial immunomodulatory proteins, which target the complement cascade at various sites, can be employed for therapeutic intervention in inflammatory responses due to activation of the complement cascade (Kotwal, G. J. (1996) Immunologist 4:157–164). For example, the inflammation modulatory protein (IMP), a small complement-binding protein that has been shown to be functionally similar to CR1, has been shown in in vivo experiments to cause a diminished specific swelling response (Miller, C. G. et al. (1997) Virology 22:126–133).
Neuroautoimmunity
Besides complement-mediated inflammation, inflammation resulting from autoimmunity has also been proposed to play a role in the pathogenesis of AD. According to the neuroautoimmunity model, cell-mediated immunity (CMI) plays a key role in the development of autoimmunity and/or inflammation (Singh, V. K. (1997) Gerontology 43:79–94). The first step in the proposed process is the formation of an autoantigen during the blood-borne acute phase, resulting in the activation of B and T cells, which give rise to anti-neuronal antibody and cytotoxic T-cell (CTL) activity, respectively. This somehow signals across the blood-brain barrier and leads to a chronic phase in the brain. In the second phase the CD8+ CTLs, either directly through CMI or indirectly through glia activation (microglial and astroglial cells), induce target cell cytotoxicity. The activation of the glial cells then causes nonspecific tissue damage. The neuron-specific degeneration characteristic of AD is due to the CMI directed by specific autoantibodies. A large number of autoantibodies and immune abnormalities, some of which are inherited, have been cataloged by Singh (id.)
Prior Modes of Treatment of Alzheimer's Disease
A major approach to the treatment of AD has involved attempts to augment the cholinergic function of the brain. An early approach was the use of precursors of acetylcholine synthesis, such as choline chloride and phosphatidyl choline (lecithin). Although these supplements are generally well tolerated, randomized trials have failed to demonstrate any clinically significant efficacy. Direct intracerebroventricular injection of cholinergic agonists such as bethanacol appears to have some beneficial effects, although this requires surgical implantation of a reservoir connecting to the subarachnoid space and is too cumbersome and intrusive for practical use. A somewhat more successful strategy has been the use of inhibitors of acetylcholinesterase (AChE), the catabolic enzyme for acetylcholine. Physostigmine, a rapidly acting, reversible AChE inhibitor, produces improved responses in animal models of learning, and in patients with AD some studies have demonstrated mild transitory improvement in memory following physostigmine treatment. The use of physostigmine has been limited because of its short half-life and tendency to produce symptoms of systemic cholinergic excess at therapeutic doses.
Recently, the acridine derivative tacrine (COGNEX®, 1,2,3,4-tetrahydro-9-aminoacridine) has been approved by the United States Food and Drug Administration for the treatment of dementia in AD. Tacrine was first synthesized nearly fifty years ago, and the pharmacology of this agent has been the subject of numerous studies. It is a potent centrally acting inhibitor of AChE. The side effects of tacrine may be significant and dose-limiting: abdominal cramping, nausea, vomiting, and diarrhea are observed in up to one-third of patients receiving therapeutic doses. Tacrine may also cause hepatotoxicity, as evidenced by the elevation of serum transaminases observed in up to 20% of patients treated. Because of the relatively small improvements that result from tacrine treatment and the significant side-effect profile, its clinical usefulness is limited.
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of treating Alzheimer's Disease, it should be apparent that there still exists a need in the art for an safe and effective treatment for AD.